Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast saccharomyces cerevisiae

Jihui Ren, Justin Snider, Michael V. Airola, Aaron Zhong, Nadia A. Rana, Lina M. Obeid, Yusuf A. Hannun

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl- CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast Saccharomyces cerevisiae. An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin Ki for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine ?-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH3 + base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.-Ren, J., J. Snider, M. V. Airola, A. Zhong, N. A. Rana, L. M. Obeid, and Y. A. Hannun. Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/ MS to study SPT activity in yeast Saccharomyces cerevisiae.

Original languageEnglish (US)
Pages (from-to)162-170
Number of pages9
JournalJournal of Lipid Research
Volume59
Issue number1
DOIs
StatePublished - 2018
Externally publishedYes

Keywords

  • High-performance liquid chromatography-electrospray ionization-Tandem mass spectrometry
  • Serine palmitoyltransferase
  • Sphingolipids

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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